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Abstract

Background

Alpha-Mannosidosis is a rare lysosomal storage disorder, caused by the deficiency
of the enzyme alpha-Mannosidase. Clinically it is characterized by hearing impairment,
skeletal and neurological abnormalities and mental retardation. In order to characterize
the clinical features and disease progression of patients affected by alpha-Mannosidosis,
a survey study was conducted. 43 patients from 4 European countries participated in
this longitudinal study. Age range of the participants was 3 to 42 years. For each
patient a medical history, complete physical and neurological examination, joint range
of motion and assessment of physical endurance and of lung function were completed.
In addition, serum and urinary oligosaccharide levels were analysed.

Methods

In this multicenter longitudinal study clinical data of 43 alpha-Mannosidosis patients
were collected. In addition to objective clinical measurements biochemical assays
were performed.

Results

Data analysis revealed a wide spectrum of clinical presentation regarding the severity
and disease progression. Most clinical abnormalities were observed in the musculoskeletal
and neurological system. All patients showed mental retardation and hearing loss from
early childhood. An impairment in physical endurance was revealed by the 6-minute
walk and 3-minute stair stair climb tests. There was only slight progression of a
few clinical findings: Psychiatric troubles in both groups essentially, and respiratory
dysfunction under 18 years. The serum and urinary oligosaccharide levels were increased
in all affected individuals and correlated well with the 6-minute walk and 3-minute
stair climb test results.

Conclusions

This study confirms that alpha-Mannosidosis is a very heterogeneous disorder regarding
both, disease severity and progression. As it has been shown that Mannosidosis patients
are able to perform lung function tests and the 6MWT and stair-climb test, these clinical
parameters apparently can be used as clinical endpoints for clinical trials. Oligosaccharide
levels appeared correlated with functional testing and may serve as biomarkers of
disease severity, progression and response to treatment.

Trial registration

Keywords:

Background

Alpha-mannosidosis (OMIM 248500) is a rare lysosomal storage disorder caused by the
deficiency of alpha-Mannosidase (MAN2B1, EC 3.2.1.24), a lysosomal enzyme responsible
for the degradation of N-linked oligosaccharides. Alpha-mannosidosis is a progressive
disorder, and characteristic features include mental retardation, coarse facial appearance,
hearing loss, skeletal deformities, central nervous system involvement and immune
defects. Based on severity two distinct phenotypes of alpha-Mannosidosis have been
described: A severe form (type I) with hepatomegaly and early death caused by severe
infections
[1], and an attenuated (mild) form (type II) with hearing loss, mental retardation and
slow progression with survival into adulthood
[2]. A further classification into a mild, moderate and severe type of alpha-Mannosidosis
seems to be questionable as the clinical presentation varies considerably
[3]. In this paper the term “severe form” (type I) and “mild form” (type II) are used.

Type I alpha-Mannosidosis leads to early death, primarily caused by the involvement
of the central nervous system and recurrent infections. In patients affected by the
mild form first symptoms such as hearing loss and skeletal abnormalities are often
seen before the age of 10 years; later ataxia and mental retardation become more and
more evident. In alpha-Mannosidosis broad heterogeneity is seen not only in the clinical
manifestations, but also in the spectrum of mutations of the alpha-Mannosidase gene
(MAN2B1) that is located on chromosome 19p13.2. Currently, 125 different disease-causing
mutations have been identified
[4]. The prevalence of alpha-Mannosidosis is not exactly known, Meikle et al. have calculated
a prevalence of 1 case in 1 million live births
[5], a similar prevalence was observed in the Netherlands
[6].

There are many reviews extensively describing the clinical manifestation of the lysosomal
storage disorder alpha-Mannosidosis; a longitudinal study, however, evaluating the
progression of the disease in a greater number of patients is lacking. Furthermore,
the clinical reports that have been published so far have been based on a small number
of patients and have not included quantitative measurements of clinical features.
Therefore, the EU consortium HUE-MAN (Towards the Development of an Effective Enzyme
Replacement Therapy for Human Alpha-Mannosidosis) was established in order to conduct
a survey of the natural history study of the human disease alpha-Mannosidosis
[7]. The purpose of this study was also to define possible clinical endpoints for future
clinical trials. In addition, clinical phenotypes of selected European patients have
been determined and important medical data such as the results of clinical and neurological
investigations, ophthalmological and hearing examinations, lung and heart function
and measurement of general endurance using the 6-minute walk test (6MWT) and 3-minute
stair climb test (3MSCT) were collected.

Methods

Clinical evaluation

The aim of this multicenter, multinational prospective study was to evaluate clinical
and surrogate parameters known to be affected in alpha-Mannosidosis patients. The
design of this observational study was open, non randomized with several objective
clinical measurements, laboratory measurements and some investigator evaluated parameters.
In addition, some patient assessed measurements (Quality of Life and Health Questionnaires)
were collected. Informed consent was obtained prior to any study related activity
and the subject, or the subject’s legal guardian, provided consent using the Ethics
Committee (EC)-approved consent form and with compliance to local and European Union
regulations, International Conference on Harmonization (ICH) and Good Clinical Practice
(GCP) standards.

Only patients with the mild form (type II) were entered into this study if they had
a documented deficiency of serum or leukocyte acid alpha-Mannosidase enzyme activity
level. Patients had to be excluded from the study if they had received a bone marrow
transplantation or had used an investigational drug within 30 days prior to study
enrollment. Known medical conditions or serious intercurrent illness that could significantly
interfere with study compliance were further exclusion criteria. In all subjects,
clinical symptoms (with special emphasis on hearing difficulties and visual impairment)
and history of infections, surgical procedures, hospitalizations and use of medication
were recorded. The physical examination included vital signs, height and weight, evaluation
for heart murmur, and for liver and spleen size.

As a measure of general endurance the 6-minute walk test (6MWT) was conducted at baseline
and at visit 12 and 24 months
[8]. The values are given as LOCF = Last Observation Carried Forward, that means that for patients who have missing data, the last previous data point
which was actually recorded is carried forward. The purpose of this is to get a mean
value which is not influenced by the large fluctuations in the means that may occur
because of the different levels for the patients. Because there are no published normal
values of the 6MWT covering the full age range, the published references were combined
in order to overcome this difficulty. For this purpose, a predictive model of the
6MWT outcomes was derived from the models published by Geiger et al.
[9], Gibbons et al.
[10] and Enright et al.
[11]. For the late adolescence, an obvious adjustment to the Geiger model was introduced.
In order to join the three models, linear interpolation was applied where the age
ranges of the models met. This procedure resulted in a combined predictive 6MWT model
that could be used across the full age range. Sensitivity analyses confirmed the suitability
of the proposed model.

In the 3-minute stair climb that was conceived on the basis of a combination of published
tests
[12,13], patients were instructed to climb as many steps as possible in a 3-minute period.

Joint range of motion encompassing passive flexion and extension at the shoulders,
elbows, and knees was measured with a goniometer utilizing standard technique. For
assessment of heart function a standard 12-lead electrocardiogram (ECG) was performed.
Interpretation of the ECG included assessment of heart rate, cardiac rhythm, intervals,
axis and conduction defects. Echocardiograms were optional, interpretation included
assessment of the size of the chambers and pumping function.

Forced vital capacity (FVC), forced expiratory volume (FEV1) and peak flow were measured
in accordance with American Thoracic Society (ATS) standards
[14]. The range of FVC was expressed as percentage of expected depending on age, size
and sex.

Ophthalmological assessments included slit lamp examination of the cornea and lens,
best corrected visual acuity (in dp) and examination of the retina of both eyes at
baseline, at 12 month and at 24 months. All three were evaluated and assessed as Normal or Abnormal and the abnormal were classified as Non Clinically Significant or Clinically significant for each eye.

Hearing was tested by pure-tone audiometry for air and bone conduction in the conventional
frequency range and for air-conduction in the extended high frequency range.

For assessment of activities of daily living, severity of pain, and extent of disability
the Health Assessment Questionnaire (HAQ) was used for subjects older than 18 years
of age
[15]. For subjects ≤ 18 years of age the Childhood Health Assessment Questionnaire (CHAQ)
was completed by the caregivers
[16].

Oligosaccharide analysis

Serum

Serum samples used in this study were stored at −20°C. Control serum samples were
obtained from healthy volunteers within the department.

250 μL of serum were mixed with 1 μg of a disaccharide (Man(β1-4)GlcNAc) which served
as an internal standard. The glycans were purified on a C18-Sep-Pak (Waters Ltd) and
on a column of 150 mg of nonporous graphitized carbon (Alltech, Deerfield, IL, USA).
After conditioning, the C18-Sep-Pak by sequential washing with methanol (5 mL), and
5% acetic acid (10 mL), the sample was loaded onto the Sep-Pak and the glycans were
eluted with 3 mL of 5% acetic acid. The glycans were then desalted on a column of
150 mg of nonporous graphitized carbon (Alltech, Deerfield, IL, USA). The column was
sequentially washed with 5 mL methanol and 10 mL 0.1% v/v TFA. The glycans were applied
to the column and washed with 15 mL of 0.1% v/v TFA. The elution of the glycans was
conducted with the application of 5 mL of 25% v/v acetonitrile in water containing
0.1% v/v TFA. The fractions were freeze-dried.

Glycans were derivatized with 2-aminobenzamide as previously described, with minor
modifications
[17]. The freeze-dried glycans were dissolved in 100 μL of a solution (freshly prepared
by mixing 64 mg of sodium cyanoborohydride, 41 mg of 2-aminobenzamide, 700 μl of dimethylsulfoxide,
and 300 μl of acetic acid). The reaction mixture was stirred for 2 h at 80°C. To remove
the excess of reagents, 500 μL of 75 and 85% methanol was added in succession to the
reaction mixture and evaporated. After addition of 2 mL of water, the pH of the solution
was adjusted to 10 with diluted ammonia solution and the excess of reagents was extracted
with 500 μL of chloroform (five times). The aqueous phase was neutralized with dilute
acetic acid prior to lyophilization. Finally, the derivatized glycans were further
purified on a Sep-Pak C18 (Waters, Saint-Quentin en Yvelines, France). The Sep-Pak C18 was conditioned with methanol (5 mL) and water (10 mL). The derivatized glycans dissolved
in water were applied on the cartridge, washed with 15 mL of water and eluted with
3 mL of 25% acetonitrile in water. Acetonitrile was evaporated under a stream of nitrogen
and the 2-aminobenzamide derivatized glycans were freeze-dried.

The 2-aminobenzamide labeled glycans were loaded on a Shodex Asahipak NH2P-50 column
(5 μm; 4.6 × 250 mm; VWR). The mobile phases were acetonitrile (solvent A) and water
(solvent B). The column was equilibrated with 85% A. After injection, isocratic conditions
were applied for 15 min with 85% A, followed by a gradient with the following conditions:
(step 1) 85-80% A for 15 min, linear gradient; (step 2) 80-50% A for 60 min, linear
gradient; (step 3) 50% A for 20 min. The flow rate was 0.7 mL/min. 2-aminobenzamide
derivatized glycans were detected on a Dionex RF 2000 fluorescence detector at an
excitation of 350 nm and an emission wavelength of 450 nm.

Urine

Samples used in this study were stored at - 20°C. Control urine samples were obtained
from healthy volunteers within the department.

Urine (0.207 μmol creatinine equivalents) was mixed with 2.608 nmol of a disaccharide
(Man(β1-4)GlcNAc) which served as an internal standard and freeze-dried. Glycans were
derivatized with the 2-aminobenzamide as previously described, with minor modifications
[17]. The freeze-dried glycans were dissolved in 100 μL of a solution (freshly prepared
by mixing 64 mg of sodium cyanoborohydride, 41 mg of 2-aminobenzamide, 700 μl of dimethylsulfoxide,
and 300 μl of acetic acid). The reaction mixture was stirred for 2 h at 80°C. To remove
the excess of reagents, 500 μL of 75 and 85% methanol was added in succession to the
reaction mixture and evaporated. After addition of 2 mL of water, the pH of the solution
was adjusted to 10 with diluted ammonia solution and the excess of reagents was extracted
with 500 μL of chloroform (five times). The aqueous phase was neutralized with dilute
acetic acid prior to lyophilization. Finally, the derivatized glycans were further
purified on a Sep-Pak C18 (Waters, Saint-Quentin en Yvelines, France). The Sep-Pak C18 was conditioned with methanol (5 mL) and water (10 mL). The derivatized glycans dissolved
in water were applied on the cartridge, washed with 15 mL of water and eluted with
3 mL of 25% acetonitrile in water. Acetonitrile was evaporated under a stream of nitrogen
and the 2-aminobenzamide derivatized glycans were freeze-dried.

The 2-aminobenzamide labeled glycans were loaded on a Shodex Asahipak NH2P-50 column
(5 μm; 4.6 × 250 mm; VWR). The mobile phases were acetonitrile (solvent A) and water
(solvent B). The column was equilibrated with 85% A. After injection, isocratic conditions
were applied for 15 min with 85% A, followed by a gradient with the following conditions:
(step 1) 85-80% A for 15 min, linear gradient; (step 2) 80-50% A for 60 min, linear
gradient; (step 3) 50% A for 20 min. The flow rate was 0.7 mL/min. 2-aminobenzamide
derivatized glycans were detected on a Dionex RF 2000 fluorescence detector at an
excitation of 350 nm and an emission wavelength of 450 nm. The mass of these glycans
were determined using MALDI-TOF-MS.

MALDI-MS experiments were carried out on Voyager Elite DE-STR Pro instrument (PersSeptive
Biosystem, Framingham, MA) equipped with a pulsed nitrogen laser (337 nm) and a gridless
delayed extraction ion source. The spectrometer was operated in positive reflectron
mode by delayed extraction with an accelerating voltage of 20 kV and a pulse delay
time of 200 nsec and a grid voltage of 66%. All spectra shown represent accumulated
spectra obtained by 400–500 laser shots. Sample was prepared by mixing a 1 μL aliquot
(5–10 pmoles) with 1 μL of matrix solution on the MALDI sample plate. The matrix solution
was prepared by saturating methanol–water (1:1) with 2,5 dihydroxybenzoic acid (DHB)
(10 mg/mL).

Results

Demographics and body measurements

Fourty five patients from 4 centers (Children’s Hospital, University of Mainz, Germany;
Department of Medicine, University Hospital of Tromsoe, Norway; Dept. of Genetic Medicine,
St. Mary’s Hospital, Manchester UK; and the Department of Pediatrics, Charles University,
Prague, Czech Republic) could be screened for this study; 31 were males and 14 were
females. The age range was 1.4 – 42.1 years, mean age was 19.8 years. 22 were under
the age of 18 years and 23 patients 18 years or older at the time of enrolment. 43
patients completed the 2 years, two children discontinued because of non-compliance.

Figures
1 and
2 show the absolute height of males and females at baseline, compared to published
growth curves for normal individuals from Northern European countries
[18]. Only 4 children (3 boys and 1 girl) had an height below the curve of the double
standard deviation. Mean height of the adult patients was 162 ± 9 cm SD with a broad
range from 145 to 179 cm.

Most of the patients in the younger age have an overall normal BMI, there are two
girls, 16 and 17 years old, who are overweight (BMI = 28.2 and 25.4, respectively).
In the adult group five patients (3 males and 2 females) are overweight with a BMI
greater than 24.0; furthermore, in this group there are two females (age 27 and 32)
who show severe obesity with a BMI of 39 and 33, respectively.

Clinical assessments

All patients had a physical examination performed at baseline, 12 months and at 24
months. Due to an unexpected early termination of the study several patients could
not complete visit 3 after 24 months.

In the general examination, all patients (children and adults) had any abnormal clinical
findings such as coarse facial features (13 of 43), ear infections (8 of 43), heart
murmur (9 of 43) and skeletal deformities such as kyphoscoliosis and pectus carinatum.

Data from the ophthalmological investigations showed that 2 patients (age 14 and 22
years) had corneal clouding, 2 patients (age 39 and 30 years) had a cataract and 2
patients (age 15 and 34 years) were almost blind (vision = 6/95 at the better-seeing
eye) due to retinal pigmentary degeneration. In one patient (25 years) a pale papilla
was seen.

From Table
1 it can be seen that the baseline visual acuity is approximately the same for both
age groups. Over the observation period, there is only a insignificant deterioration
in visual acuity.

Table 1.Visual acuity (best corrected, best seeing eye) of both age groups

All patients who were able to cooperate had an assessment of their hearing performed
at baseline, at 12 months and at 24 months. The assessment included: Bone conduction,
air conduction (conventional frequency) and air conduction (high frequency). All patients
over the age of 3 years had a significant hearing loss and had to wear hearing aids.
Data of the hearing tests, expressed as decibel, show that baseline values are quite
similar between the two age groups (Figures
4,
5,
6).

The data also seem to indicate a minor change of hearing loss over the first part
of the observation period which also is similar between the groups. For the second
part of the observation period from 12 to 24 months there seems to be a slight improvement
in hearing. Missing data limits a more detailed interpretation of the results.

For the cardiovascular system 24% of the patients under the age 18 years and 17% of
patients age 18 or over presented abnormal findings. In the majority of cases they
were clinically not significant as exemplified as “murmur” on cardiac auscultation.
Five patients had an abnormal echocardiogram: The findings were given as valvular
insufficiency or decreased ventricular ejection fraction. Two patients (one in each
age group) had an abnormal ECG that was assessed as clinically significant; more findings
were not reported.

The musculoskeletal area showed typical abnormal findings for this disease such as
macrocephaly, contractures, scoliosis, genua valga, hip dysplasia and deformities
of feet. In the younger age group (patients < 18 years) 62% of the patients had abnormal
findings for this body area. It seems to be stable over the observation period, which
would also be expected. In the older patients pathological signs and symptoms of the
musculoskeletal system were observed at an even higher rate than in the younger age
group: In this population 92% of the patients had abnormal findings for this body
area at baseline. After two years 94% of adult patients had abnormal findings.

Neurologically 71% of the 20 patients under the age of 18 years showed abnormal signs
and symptoms at baseline, whereby the main clinical sign was ataxia that was seen
in 12 of the patients. Further findings included dysarthria, dysmetria, and mental
retardation. A similar rate of pathological neurological findings (79%) was seen in
the older patients. Over the observation period this did not change significantly,
in either the younger or older age group.

Psychologically, 52% of the patients had abnormal findings at baseline. Despite a
decrease at visit 2 in the percentage of patients with abnormal findings (e.g. psychotic
disorder) there was a significant increase at visit 3 with 79% of the patients showing
abnormal findings.

6-minute walk test and 3-minute stair climb test

All patients were expected to perform a 6-minute walk test (6MWT) at baseline, at
12 months and at 24 months. However, as the youngest patient was not cooperating,
only 19 of 20 patients under the age of 18 carried out the test at baseline. Following
amendment 1 some patients did not have the opportunity to complete either the amended
18 months test (due to the timing of the amendment) or the 24 months test (because
of the early stop of the study). Hence the number of patients completing the projected
tests differ considerably between time points.

As shown in Table
2 the distance that could be walked within 6 minutes at baseline ranged from 60,0 to
470,0 m with a mean value of 341,1m (SD = 104,9 m) in the younger age group, and from
69,0 m to 521,3 m with a mean value of 324,7 m (SD = 118,1 m) in the older group.

In order to compare the outcome of the 6MWT in the patient group with normal subjects,
the predictive model of the 6MWT outcomes, published by Geiger et al.
[9], Gibbons et al.
[10] and Enright et al.
[11], was used (see Methods). Calculations by using the Geiger model show that for the
patients in the younger patient population the functional capacity is reduced to a
value between 50 and 60% of the values for normal patients of the same age (Figure
7). For the older age group (above 18 years of age) the functional capacity for a 6
MWT is further reduced to less than 50% of the functional capacity for normal patients
(ie 40 – 45%).

At baseline, the number of stairs that could be climbed was 92–166 stairs with a mean
value of 141 (SD = 30) stairs in the younger age group, after 1 year a slight improvement
was observed (106–197 stairs, mean value 160, SD = 39 stairs). Patients at the age
over 18 years could climb at baseline 22–186 stairs (mean value = 100, SD = 58), they
also showed an improvement after one year (43–197 stairs, mean value = 126, SD = 55
stairs). Data of the 2 years visit are not available.

Lung function tests

Eleven of patients under the age of 18 years and 9 adult patients were able to perform
a pulmonary function test at baseline, at 12 months, at 18 months and at 24 months.
The assessments included: Forced vital capacity (and percentage of predicted value,
depending on age, size and sex = % predicted FVC), forced expiratory volume during
first second (and percentage of predicted value = % predicted FEV1) and peak expiratory
flow rate. In all measurements there was a significant range, spanning from about
30% to 90% of predicted FVC and FEV1. No patient was dependent on night time ventilation.

In patients under the age of 18 the % predicted FVC is decreasing from a baseline
value of 71% to 61% at visit 3 (Figure
8) that clinically is not significant.

For the older age group the overall picture of the pulmonary function capacity is
similar over time; for the younger age group the data seem to indicate that over the
observation period there is up to a 10% decrease in pulmonary function, that clinically
is not significant.

Cardiology assessment

All patients had an assessment of their ECG and an overall interpretation of an echocardiogram
at baseline, at 12 months and at 24 months. The ECG and echocardiogram were evaluated
and assessed as Normal or Abnormal and the abnormal results were further classified as Non Clinically Significant or Clinically significant. There are two patients in the older age group and one in the younger age group who
present with clinically significant abnormal findings. The findings are given as valvular
insufficiency or stenosis or decreased ventricular ejection fraction. These abnormal
findings do not change over the observation period.

Oligosaccharides in serum and urine

Valid serum oligosaccharide levels were available for 5 subjects under the age of
18 years and from 11 individuals over 18 years. The older patients had higher concentrations
(11.05 – 82,45 nmol/ml, mean = 21,49 nmol/ml) than the younger patients (7.16 – 33.49
nmol/ml, mean = 16.33 nmol/ml). In the urine, the oligosaccharide concentration was
similar in both age groups, whereby the mean value at the age under 18 years (n =
10) was 323.00 nmol/ml and in adults (n = 15) 280.62 nmol/ml. The 6MWT as an indicator
of general endurance was examined as a function of oligosaccharide urine/serum. And
as can be seen in Figure
10, low oligosaccharide levels corresponded to a long walking distance and vice versa.
Similar results were obtained when the analysis was applied to the 3 minute stair
climb test (Figure
11).

The health assessment questionnaires

The health assessment questionnaires confirm that the patients are dependent to a
high degree upon third party assistance. This is not age dependent as it is seen in
both age groups. A major difference is the pain score: The VAS scores clearly show
that the older age group seem to have more pain than the younger age group (as far
as the mentally retarded children could give faithful responses). The reason for this
is not clear, but it is likely to represent progressive bone and joint disease.

Discussion

In the present study that was aimed to establish the range and diversity of clinical
features of alpha-Mannosidosis and to define clinical endpoints for future clinical
trials, information was collected for 43 affected subjects over the age of 3 years,
identified by four centers from four European countries. A total of 45 patients were
screened; during the course of the study, however, two patients discontinued because
of no-complicance. In addition to clinical investigations, analyses of oligosaccharide
concentration in urine and serum and were performed.

This longitudinal survey was also conducted in order to assess the natural history
of patients diagnosed with alpha-Mannosidosis and to evaluate short term (24 months)
changes in disease parameters. Previously only a few retrospective studies describing
the clinical course have been published and these have included only a small number
of patients: Yunis et al. analyzed the clinical course of five patients who in their
age ranged from 15 to 24 years
[19]. The authors pointed out that the first signs and symptoms such as hearing problems,
skeletal changes and facial coarsening could be seen from the age of five years; the
degree of clinical expression, however, varied from patient to patient, even among
siblings. In the brother and sister, described by Ara et al., at the age of 27 and
29 years, respectively, neurological changes such as cerebellar syndrome, hearing
loss and mental retardation were the leading clinical signs; except in the sisters’s
speech capacity, over a period time of 25 years no progression was observed
[20]. Neurodevelopmental assessments including general intelligence, language, visual
spatial skills and overall adaptive abilities were performed in three brothers with
alpha-Mannosidosis; follow-up studies did not show signs of progressive deterioration
of cognitive deficits, except receptive language capability
[21].

The data from the physical examination confirm that alpha-Mannosidosis is a complex
disorder affecting many organs such as the eyes, ears and the central nervous and
the musculoskeletal system. Abnormalities of the musculoskeletal system are one of
the clinical findings that affect almost all patients. Whereas in patients under the
age of 18 years skeletal deformities were seen in 62%, scoliosis, genua valga, hip
dysplasia and joint contractures have been observed in almost all adult patients (92%).
A similar frequency of skeletal changes (55 of 59 patients) was described in the review
of Chester et al.
[22]. A lessening of the bone abnormalities, as described by Yunis et al.
[19] could not be confirmed in our study; the severity of of bone deformities did not
change over the time. In contrast to the observations of Autio et al.
[23] and Yunis et al.
[19] a noteworthy growth retardation was not seen in all patients in spite of significant
skeletal abnormalities: Only four children had an height below the normal range, in
the adults a broad range of height from 145–179 cm was seen. Only a few patients have
a BMI above the normal range.

The joint range was measured rigorously, but in a non functional manner, and despite
many abnormal findings in the physical examination for the joints, there seemed to
be no deterioration in range of joint movement over the observation period.

Ataxia and mental retardation were the prominent neurological findings observed in
this study; a progression of these symptoms within 2 years could not be seen. A similar
observation was made by Autio et al. who described a merely slow progression of mental
retardation in eight patients. These patients had an IQ of 60–80, whereby they scored
better in nonverbal tests which can be explained by the speech disturbance due to
the hearing impairment
[23].

Hearing loss (bone and conductive) is seen in all patients affected by an alpha-Mannosidosis
[22,24]. The impaired hearing is predominantly due to sensorineural damage, but because it
may result also from early ear infections it represents a mixture of conductive and
neurosensory components as it has been seen in this study. As hearing loss appears
during early childhood, it is not surprising that in our study both age groups show
more or less the same degree of hearing impairment. A significant deterioration within
the observational time could not be seen.

Ophthalmological investigations in most patients revealed only minor abnormalities
such as slight corneal opacities or cataract and amblyopia. These results are in accordance
with the observations of other authors who also have described strabismus, hypermetropia
and slight corneal clouding in only a few cases
[23,25]. Retinal pigmentary degeneration, leading almost to blinding, has been found in two
of our patients; a similar observation was made by Springer et al. in two brothers
[26]. In general, however, retinal dystrophy seems to be rare in Mannosidosis patients.
There were no significant changes of the ophthalmological findings in our study throughout
the observation period.

Besides innocent heart murmur the majority did not have significant cardiac abnormalities.
In five patients, however, valvular insufficiency or decreased ventricular ejection
fraction was seen by echocardiogram. Two patients had an abnormal ECG that was not
specified in detail by the investigator.

Because to our knowledge there is a paucity of data on respiratory function in Mannosidosis
patients, lung function tests were performed in the present study. However, not all
patients were able to perform the lung function tests because of their mental status.
In the younger age group there was a decline of percentage predicted FVC within the
observation time of 2 years. The older patients had a lower percentage predicted FVC
(61%) at baseline in comparison with the children (Figure
8).

The 6 MWT has not previously been performed in Mannosidosis patients, therefore this
method was used to measure the general endurance of these individuals in the present
study. However, both the actual distance measured as well as the percentage of normal
distance does not show any consistent picture either in the younger or in the older
patient age group. Both groups show a significant range, spanning from about 60 to
about 500 meters. A number of factors such as parental influence, growth or a training
effect may have influenced the 6MWT. A similar consideration applies to the 3 minute
stair climb test. There is no clear trend throughout the observation period for either
age group probably for similar reasons as stated for the 6 MWT.

Mannosidosis patients excrete high amounts of undegraded oligosacharides that can
be detected in their urine. Serum samples showed similar findings. The storage material
is composed mainly of tri-, tetra-, and pentasaccharides and probably represents the
products of endo-ß-N-acetylglucosaminidase digestion of the sugar chains of high mannose
type glycoproteins
[27,28]. In our study, an interesting correlation of the 6MWT and oligosaccharide excretion
could be observed in both age groups: Patients with a high oligosaccharide excretion
have an impaired ability to walk (Figure
10). The same correlation was found of oligosaccharide excretion and the stair climb
test (Figure
11). As a relationship of general endurance and urinary oligosaccharide levels was seen
in all age groups, it can be presumed that the oligosaccharide excretion rather depends
from the severity of the disease than from the age of the patients. In patients affected
by mucopolysaccharidosis type VI a similar observation was made: MPS VI patients who
had low height values excreted higher amounts of glycosaminoglycans than those who
had normal or almost normal height values
[29]. From these findings it can be concluded that urinary oligosaccharide excretion can
be used as a surrogate marker in clinical trials for example for the development of
therapeutic principles such as enzyme replacement therapy.

Conclusions

The variable clinical presentation of the Mannosidosis patients investigated in this
survey confirms that alpha-Mannosidosis is a highly heterogeneous disorder, as it
has already been described by several authors
[1,3,22]. It could be demonstrated that there was only slight progression of a few clinical
findings within the observational time: In both groups psychiatric troubles, and impaired
lung function in patients under the age of 18 years. Our study has revealed that in
both age groups significant neurological abnormalities such as ataxia and dysarthria
can be observed. And in a mouse model for alpha-Mannosidosis it could be demonstrated
that the intravenous administration of recombinant alpha-Mannosidase resulted in an
improvement of the neuromotor disabilities found in untreated mice
[30]. From these results it can be considered to incorporate not only the lung function
and 6MWT or 3stair climb test, but also neurological investigations as clinical parameters
for therapeutic clinical trials
[31]. As additional biomarkers oligosaccharide levels in serum and/or urine may serve.

Abbreviations

Competing interests

The study is funded by the EU in the “HUE-MAN” consortium through a Framework 6 grant
of 3.2 million Euro for a period of 36 months. The entire grant covers both scientific
programs and clinical programs. The authors; except Jens Fogh, who is employed at
the Danish company Zymenex; confirms independence from any sponsors; the content of
the article has not been influenced by any sponsors.

Authors’ contributions

DM has designed the study and has evaluated patients. PS and JF are the coordinators
of the project. MB, JZ and JEW have performed the clinical evaluations of the patients.
JCM has performed the biochemical analyses. KJO has carried out the statistical analysis.
All authors contributed to the concept of the study and participated in drafting the
manuscript and approved the final manuscript.

Acknowledgements

This work was supported by the HUE-MAN consortium (European Commission FP VI contract
LHSM-CT-2006-018692). It is written in memory of Dr. Ed Wraith who deceased during
the preparation of the manuscript. Dr. Ed Wraith made significant contributions to
research and improvements in the care of patients affected by a lysosomal storage
disorder. He will be fondly remembered by colleagues for his enormous scientific work
and his kindness, and by patients for his excellent clinical skills and compassion.